| Research Abstract |
The sorting of isoproteins of myosin alkali light chain (LC) and troponin I (TnI) during myofibril assembly was analyzed by force-expression of epitope-tagged cDNAs in cultured cardiac and skeletal muscle cells and fibroblasts. In cardiomyocytes possessing cardiac myosin heavy chain (MHC), sorting specificity of LC isoforms to the sarcomeres was increased in the order from nonmuscle LC3nm, to slow muscle LC1sa, to slow/ventricular muscle LC1sb, and to fast muscle LC1f and LC3f. In fibroblasts expressing nonmuscIe MHC, the order of LC1sa and LC1sb in cardiomyocytes was reversed. Thus, the sorting of LC occurred depending on MHC isoforms as well as LO isoforms. Analysis of subcellular distribution of force-expressed chimeric LCs revealed that the second EF-hand of LC is responsible for such isoform specific sorting. Force-expressed cardiac and fast muscle TnI were incorporated into myofibrils of cardiac and skeletal muscles, respectively. However, cardiac TnI was not incorporated into sk
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eletal muscle myofibrils, although fast muscle TnI was assembled on to cardiac myofibrils. Expression of chimeric TnI revealed that the differential behavior of TnI isoforms depends on difference in the C-terminal halves of the isoproteins. The exchangeability of actin was examined by injection of rhodamine-labeled actin and fluorescence recovery (FR) after photobleaching in cardiomyocytes and fibroblasts. The FR rate in stress fibers was consistently faster than that in myofibrils, indicating that stress fibers and non-striated myofibrils are different in actin stability, although they are similar in appearance and composition. In myofibrils, non-striated parts exhibited faster FR rate than striated portions, and the FR rate of muscle actin was faster than that of nonmucle type. Incorporation of LC3f fused with green fluorescence protein (LC3f-GFP) into myofibrils was monitored in living cardiomyocytes. LC3f-GFP was initially accumulated at both ends of A-bands, and then incorporated into the whole of A-bands except the H-zone. Less
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